2012  •   Computer and Information Sciences

Resuscitation of HipA-mediated persistence

Lead Presenter: Alessandro Calo


Computer and Information Sciences

Bacterial populations stochastically produce a small number of non-growing or dormant persister cells that are tolerant to antibiotics. Persisters are phenotypic variants that are genetically identical to the susceptible cells within a clonal population. Thus, persistence is a non-inheritable, transient trait. A targeted search for mutants of E. coli with high levels of persistence led to the finding of a strain with two point mutations in hipA (hipA7 allele) that produced up to 10,000 times more persisters than the wild type. HipA is part of a two gene operon, hipBA, which encodes a toxin/antitoxin pair. HipA leads to growth arrest and high persistence upon overproduction. Persisters resume growth after an external stress factor is removed, ensuring survival of the population. Resuscitation of persisters is poorly understood. To identify a resuscitation factor of HipA, we designed a screen based on growth arrest associated with HipA overexpression. We transformed a pool of the ASKA library (pCA24N) into KLE969, which has arabinose-inducible pBADhipA inserted into ?attB, and selected clones that produced colonies in the presence of the inducer. 130 clones were initially selected. 10 clones repeatedly showed full growth on arabinose/IPTG plates. The respective inserts in pCA24N were identified by sequence analysis. Among the genes counteracting HipA-mediated toxicity is one known protein phosphatase prpA, two other phophatases yigB and ycjU, as well as apaG which is in an operon with apaH, which has previously been linked to persistence. Finally, interaction of resuscitation factor candidates with HipA is being validated in pull-down studies.